1. Field of the Invention
The present invention generally relates to calibrator nucleic acid molecules. The calibrator nucleic acid molecules may be used in qualitative and quantitative nucleic acid assays such as quantitative real-time PCR assays.
2. Description of the Related Art
Quantitative real-time polymerase chain reaction (Q-PCR) is used to accurately quantitate the level of messenger RNA (mRNA) for a polynucleotide of interest in a biological sample. Currently, Q-PCR is the most sensitive and robust technique for the quantitation of mRNA and the determination of expression levels of a gene. The quantitation of mRNA by Q-PCR is determined in relation to an internal reference gene that is expressed at constant levels in a series of samples. Several internal reference genes, such as beta-actin, GAPDH, and the like, have been used in Q-PCR and are referred to as “housekeeping” genes. That is, the expression level of these genes has been thought to remain relatively constant across a sample set.
Use of housekeeping genes, however, is not always appropriate since various experimental conditions have been shown to alter the levels of housekeeping genes. In these situations, a known amount of a calibrator polynucleotide that is added to the sample prior to processing and analysis may be used. The calibrator polynucleotide then becomes the internal reference standard for the Q-PCR assay. The levels of the calibrator polynucleotide are independent of the experimental conditions, thereby resulting in an accurate internal standard.
The theoretical utility of a calibrator polynucleotide has been discussed previously. Bustin described the use of universal controlled reference polynucleotides to control for reverse transcriptase and PCR efficiencies. See Bustin, S. A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Molecular Endocrinology. 29:23-39, which is herein incorporated by reference. Unfortunately, as explained by Bustin, externally added calibrator polynucleotides are not widely accepted and commercially available as validated universal calibrated polynucleotides which is reiterated by Huggett et al. See Huggett et al. (2005) Real-time RT-PCR normalization; strategies and considerations. Genes and Immunity. 6:279-284.
Thus, a need still exists for universal and validated calibrator polynucleotides for quantitative and qualitative nucleic acid assays such as quantitative real-time PCR assays.